Axio imager a1 fluorescence microscope

Samples were blocked and permeabilized in 20% normal goat serum (NGS) in 0.05% Triton-X-100 in DPBS for 1.5 h at room temperature. Primary antibodies were applied for 1.5 h at room temperature or overnight at 4°C and secondary antibodies were applied for 2 h at room temperature. Nuclei were counterstained with Hoechst 33342 (1:25,000) for 10 min at room temperature. All washes were in DPBS. For full list of antibodies and concentrations see Supplementary Table 1. Samples were mounted using Vectashield mounting medium and imaged using a Zeiss Axio Imager A1 fluorescence microscope and Axiovision software (v4.0). Negative control tissues used for immunofluorescence analysis were as follows: (a) negative controls for alginate beads: CGR8 were encapsulated in alginate beads and cultured for 6 days in ESC medium with LIF to prevent differentiation, (b) HGF11-derived MN characterization: HGF11 ESCs were cultured on gelatin-coated coverslips in ESC medium until confluent, and (c) HGF11-derived MN GFP/FoxP1 validation: HGF11 ESCs were differentiated using 1 μM RA only. CGR8 ESCs were differentiated with 1 μM RA and 1.5 μM purmorphamine toward a MN lineage.

Huh7 and BNL cells were plated on 4-well chamber slides, allowed to attach overnight, and exposed to ionizing irradiation of 10 Gy or 5 Gy either alone or combined with 1 μM of BKM120. After treatment, Huh7 cells were incubated for 30 minutes, 4, 6, and 8 hours, BNL cells were incubated for 30 minutes, 1, 2, and 4 hours, washed three times with ice-cold phosphate-buffered saline (PBS), fixed in 4% formaldehyde/PBS for 30 minutes, permeabilized in 0.5% Triton X-100 in PBS for 30 minutes, blocked in 5% bovine serum albumin for 1 hour at room temperature, incubated with the antibody (fluorescein isothiocyanate [FITC]-conjugated anti-phospho-histone γH2AX [Ser139; 1:500; Millipore, Billerica, MA, USA]) for 16 hours at 4.C in the dark, washed with PBS, and mounted in Vectashield mounting medium containing diamidino-2-phenylindole (Vector Laboratories, Burlingame, CA, USA). γ-H2AX foci were examined using a Zeiss Axio Imager A1 fluorescence microscope. In each sample, the number of γ-H2AX foci per nucleus was counted using an automated foci counter under high power field, and an average of 150 nuclei were analyzed. The average number of γ-H2AX foci per nucleus represents the amount of double strand breaks (DSB).

Cells were grown overnight, subcultured in YPD broth and grown to exponential phase. Cells were harvested by centrifugation and washed twice with phosphate-buffered saline (PBS). The cells were placed in PBS buffer containing MitoSOX Red (5 μM) and incubated at 30 °C for 30 min to stain the mitochondria. Subsequently, cells were washed twice with PBS, twice with 12.5 mM sodium acetate, and then resuspended in 12.5 mM sodium acetate to 2 × 10⁶ cells/ml. Cells were treated with different concentrations of FITC-labeled AMPs (P-113, 1 µM; P-113Du, 0.1 µM; P-113Tri, 0.05 µM). The localization of the AMPs was detected using a Zeiss Axio Imager A1 fluorescence microscope (HBO100 Mercury Lamps, Filter set 56 HE: EX (GFP) BP 470/27, EX (DsRed) BP 556/25, Zeiss n-achroplan 100×/1.25 oil lens). All images were taken within 1 h at 37 °C after AMP addition. A total of 168 images were taken and analyzed with the SPOT Basic software (SPOT Imaging, Sterling Heights, USA).

Following pharmacological treatment (48 h; Control, 30 µg/ml PAN, 100 µg/ml RTX, PAN + RTX), Hoechst 33342-staining of podocytes was performed as previously described^{25 (link)}. After collecting supernatant medium with detached cells, cultures were washed with PBS and detached using Trypsin–EDTA (Biochrom, Berlin, Germany) for 5 min at 37 °C, pelleted for 5 min at 1200 rpm, washed with PBS and resuspended in 2.5 ml growth medium. Hoechst 33342 (Sigma, München, Germany) was added to the medium to a final concentration of 1 µM/ml for 15 min at 37 °C. Cells were fixed for 15 min at room temperature with 4% formaldehyde (Fischar, Saarbrücken, Germany). After pelleting, cells were resuspended in 100 µl PBS, dispensed on a microscope slide and dried for 1 h in the dark. Slides were covered with ProLong Gold Antifade Reagent (Invitrogen, Karlsruhe, Germany) and coverslips. Images were randomly obtained by a Zeiss Axio Imager A1 fluorescence microscope and Axio Vision SE64 Rel. 4.9.1 software (Zeiss, Jena, Germany). Analysis was performed with ImageJ (https://imagej.nih.gov/ij/) software. Apoptosis was defined as percentage of cells with nuclear condensation/fragmentation. Condensed/fragmented nuclei were then counted by an observer unaware of the experimental conditions. For each sample in a given experiment, at least 200 randomly chosen cells were analyzed.

The procedure of cell migration assay was followed in a previous study [25 (link)]. First, 1 × 10⁴ per well of MSCs were added into Oris seeding stoppers for 24 h of incubation to reach confluency. Next, the stoppers were removed and used as a pre-migration reference (t = 0 h). The post migration was represented at 24 h. Next, 200 μL of a Calcein AM solution (2 μM, Sigma-Aldrich, Burlington, MA, USA) was added to the culture plates and stained for 30 min at the time points of 0 and 24 h. The cells were observed by a Zeiss Axio Imager A1 fluorescence microscope (White Plains, NY, USA). Cell migration distance was semi-quantified using Image J 5.0 software (Becton Dickinson, Canton, MA, USA). The MSC alone represented as the control.

To prepare slides for nuclear staining, HL-60 cells were diluted to a concentration of 5 × 10⁵ cells/mL in 200 μL and centrifuged onto glass slides for 5 min at 500 rpm using the Shandon Cytospin 2 centrifuge (Shandon Southern Products, Cheshire, England) [1 (link)]. The attached cells were fixed with chilled methanol for 5 min and staining of nuclei was achieved by mounting the slides with DAPI-contained VECTASHIELD Vibrance Mounting Medium. An AxioImager A1 fluorescence microscope (Carl Zeiss, Toronto, ON, Canada) equipped with DAPI filter cube was used to view the slides. The images (Figure 1a) were taken with a high-resolution monochrome XCD-X700 CCD camera (Sony of Canada Ltd., Toronto, ON, Canada) using Northern Eclipse 8.0 software from Empix Imaging (Mississauga, ON, Canada) and used to calculate the differentiation index, i.e., the percentage of nuclei with non-ovoid (indented, lobulated, or multilobed) morphology representing differentiated cells versus nuclei with ovoid/rounded morphology representing undifferentiated cells [27 (link),28 (link)]. At least three randomly selected areas were imaged on each slide, the numbers of ovoid (N₁) and non-ovoid (N₂) nuclei were counted using ImageJ (11–46 cells per image), and the differentiation index was calculated as N₂/(N₁ + N₂) × 100%.